Modified 13-hydroperoxide lyases and uses thereof

ABSTRACT

Fatty acid 13-hydroperoxide lyase proteins which have been modified with respect to a previously described guava 13-hydroperoxide lyase and the nucleic acid sequences encoding these proteins. Also, recombinant nucleic acid molecules for expressing the modified 13-hydroperoxide lyases and methods of using such lyases in the field of organic synthesis.

This application is a continuation application of application Ser. No.12/664,565 which is a 371 filing of International Patent ApplicationPCT/IB2008/052539 filed Jun. 25, 2008.

FIELD OF THE INVENTION

The present invention relates to fatty acid 13-hydroperoxide lyaseproteins which have been modified with respect to a previously describedguava 13-hydroperoxide lyase and the nucleic acid sequences encodingthese proteins. The present invention also relates to means forexpressing the modified 13-hydroperoxide lyases and methods of usingsuch lyases in the field of organic synthesis.

BACKGROUND ART

Amongst the compounds which are useful in the perfume and flavorindustry, involving hydroperoxide cleavage possibly carried out viaenzymatic reaction, the so called “green notes” include n-hexanal,hexan-1-ol, 2-(E)-hexen-1-al, 2-(E)-hexen-1-ol, 3-(Z)-hexen-1-ol (alsoknown as pipol) and 3-(Z)-hexen-1-al, which are widely used in flavors,particularly fruit flavors, to impart a fresh green character.Furthermore, green notes are essential for fruit aroma and are usedextensively in the aroma industry. The demand for natural green noteshas grown to exceed their supply from traditional sources such as mint(Mentha arvensis) oil. This has motivated research efforts towardfinding alternative natural ways of obtaining these materials.

The synthesis of green note compounds starts from free (polyunsaturated)fatty acids such as linoleic (9-(Z), 12-(Z)-octadecadienoic) anda-linolenic (9-(Z), 12-(Z), 15-(Z)-octadecatrienoic) acids. In nature,these acids are released from cell membranes by lipolytic enzymes aftercell damage. Fatty acid 13-hydroperoxides are formed by the action of aspecific 13-lipoxygenase (13-LOX) and are subsequently cleaved by aspecific 13-hydroperoxide lyase (13-HPOL) into a C₆ aldehyde and a C₁₂ω-oxoacid moiety. The aldehydes can subsequently undergo thermalisomerization and/or be reduced by dehydrogenase enzymes to produceother C₆ products (i.e., green notes) such as alcohols (Hatanaka A.(1993) Phytochemistry 34: 1201-1218; Hatanaka A. et al. (1987) Chemistryand Physics of Lipids 44: 431-361).

Guava has been identified as an excellent source of freeze-stable13-HPOL for use in this synthetic pathway. Guava 13-HPOL is currentlyused in an industrial process for the production of green notes (U.S.Pat. No. 5,464,761). In this process, a solution of the required13-hydroperoxides is made from linoleic or linolenic acid (obtained fromsunflower and linseed oils, respectively) using freshly prepared soybeanflour as a source of 13-LOX. This solution is then mixed with a freshlyprepared puree of whole guava (Psidium guajava) fruit as the source for13-HPOL. The aldehyde products are then isolated by distillation. Whenthe corresponding alcohols are required, fresh baker's yeast is added tothe hydroperoxide solution before it is mixed with the guava puree. Theyeast contains an active alcohol dehydrogenase enzyme that reduces thealdehydes to the corresponding alcohols as the aldehydes are formed by13-HPOL.

There are a number of disadvantages to this industrial process. Theprincipal disadvantage is the requirement of large quantities of freshguava fruit. This means that the process has to be operated in a countrywhere fresh guava fruit is cheaply and freely available. Even if such asite is found, availability is limited to the growing season of thefruit. Good quality guava fruit, for example, is only available for tenmonths of the year in Brazil.

A second disadvantage is that the desired enzyme activities are ratherdilute in the sources employed. This means that relatively large amountsof soy flour (5%), guava puree (41%) and yeast (22%) have to be used inthe process. The large volumes of these raw materials that are requiredfor industrial production place physical constraints on the yields ofgreen notes that can be achieved and raise the costs for the industrialprocess.

A third disadvantage is that it is a large-volume batch process, which,by its nature, does not make maximum use of the 13-HPOL enzyme'scatalytic activity, is relatively labor-intensive and generates a largeamount of residual organic material. The residual organic material mustsubsequently be transported to a compost farm or otherwise discarded.

To overcome some of the disadvantages of this industrial process, EP 1080 205 discloses purified and recombinant guava 13-HPOL proteins,nucleic acids, expression systems, and methods of use thereof. However,upon using the recombinant guava 13-hydroperoxide lyase for producingC₆-aldehydes, it turned out that the yield of products obtained with therecombinant guava 13-hydroperoxide lyase could still be optimized.

Thus, there is still a strong interest in providing 13-HPOL enzymeswhich allow the recombinant expression of the enzyme and which can beused to obtain a high yield of the desired product.

The above mentioned objects of the invention are solved by the modified13-hydroperoxide lyase polypeptides according to claim 1. Preferredembodiments are represented by the subject matter of the sub-claims.

SUMMARY OF THE INVENTION

The present invention provides a modified 13-hydroperoxide lyasepolypeptide which comprises an amino acid sequence which has 1 to 40amino acid alterations as compared to the amino acid sequence of thewild-type protein according to SEQ ID No. 1 and includes at least oneamino acid alteration in a position selected from the group consistingof positions 3, 4, 5, 19, 208, 340, 342, 352, 354, 358, 359, 360, 371,372, 375, 377, 382, 383, 387, 388, 389, 392, 393, 394, 395, 399 and 457of the wild-type protein according to SEQ ID No. 1.

The present invention further provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a modified 13-hydroperoxidelyase of the present invention, recombinant nucleic acid molecules whichallow the expression of the modified 13-hydroperoxide lyase in suitablehost cells and transgenic cells comprising such recombinant nucleic acidmolecules.

The present invention also provides a method for preparing a modified13-hydroperoxide lyase polypeptide of the present invention by culturingone or more transgenic cells under conditions which permit expression ofthe polypeptide and optionally recovering the polypeptide.

The present invention is further directed to the use of the modified13-hydroperoxide lyase of the present invention for cleaving a13-hydroperoxide of a polyunsaturated fatty acid into an aldehyde and anoxocarboxylic acid.

Finally, the present invention provides a method of producing analdehyde by providing a modified 13-hydroperoxide lyase according to thepresent invention, contacting a 13-hydroperoxide of a polyunsaturatedfatty acid with the modified 13-hydroperoxide lyase and recovering theproduced aldehyde or the corresponding alcohol after reduction of thealdehyde.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified 13-hydroperoxide lyasepolypeptides, nucleic acid molecules coding for said modifiedpolypeptides, means for their expression and methods using the modified13-hydroperoxide lyase.

The inventors have surprisingly found that introducing amino acidalterations into the amino acid sequence of guava 13-hydroperoxide lyaseas disclosed in EP 1 080 205 by mutagenesis methods such as DNAshuffling and error prone PCR leads to modified enzymes with improvedenzymatic activity as compared to the non-modified recombinant enzymefrom guava. Furthermore, it has been found that the recombinant hostcells which express the modified hydroperoxide lyase exhibit anexcellent storage stability, i.e. they can be stored for several months,particularly if they are kept refrigerated. Another advantage of thepresent invention is that the modified enzymes show high initialreaction rates, which allow the production of a high quantity of productwithin a short period of time while using only low amounts ofrecombinant biomass. Furthermore, it is possible to re-use the modifiedenzymes in further reactions. Finally, it has been shown that thequality of the products obtained with the modified 13-hydroperoxidelyases of the present invention is superior to the existing productswhich are currently used for example in perfumery, as almost noisomerization occurs while the product is formed.

Within the meaning of the present invention, a “lyase” means a proteinhaving at least one lyase function, i.e. the capability to catalyze thecleavage of a molecule. In particular, the term “13-hydroperoxide lyase”means that the lyase protein is capable of cleaving 13-hydroperoxides ofpolyunsaturated fatty acids. For example, the 13-hydroperoxide lyase iscapable of cleaving a fatty acid 13-hydroperoxide of linoleic acid orlinolenic acid into a C₆-aldehyde and a C₁₂-oxoacid moiety.

In terms of the invention, “modified” or “variant” means that the aminoacid sequence of the modified or variant polypeptide is altered ascompared to the amino acid sequence of the wild-type protein accordingto SEQ ID No. 1.

Modified 13-HPOL enzymes can be obtained by a person skilled in the artby different means such as DNA engineering including gene synthesis,site-directed mutagenesis, site-saturation mutagenesis and any otherdirected evolution technologies (random mutagenesis, shuffling, etc.) inwhich new DNA sequences are generated to create new variants (orlibraries of new variants). This can be done from a single gene usingrandom mutagenesis (WO 2006/003298). Diversity could also be generatedfrom several parental genes using a family shuffling recombination stepas described for instance in the WO 00/09679. Variants are then screenedin order to select under stringent conditions the improved enzymaticactivity. These improved variants can then be used in the next round ofevolution. The amino acid sequence of the modified 13-hydroperoxidelyase has 1 to 40 amino acid alterations, preferably 5 to 35, alsopreferably 4 to 25, more preferably 6 to 25, even more preferably 10 to30 and most preferably 17 to 25 amino acid alterations as compared tothe amino acid sequence of the wild-type protein according to SEQ ID No.1.

The term “wild-type protein” refers to the modified 13-hydroperoxidelyase enzyme as disclosed in EP 1 080 205, but with a deletion of 31amino acids at the N-terminus compared to the sequence disclosed in EP 1080 205. Enzymes carrying this deletion showed improved expressionleading to a higher catalytic activity as compared to enzymes that donot show this deletion. The sequence of the wild-type protein accordingto the present invention is depicted in SEQ ID No. 1.

The term “amino acid alteration”, as it is used herein, is intended tocomprise an insertion of one or more amino acids between two aminoacids, a deletion of one or more amino acids or a substitution of one ormore amino acids with one or more different amino acids, as compared tothe amino acid sequence of the wild-type protein as depicted in SEQ IDNo. 1. The amino acid alterations can be easily identified by comparisonof the amino acid sequence of the modified protein with the amino acidsequence of the wild-type protein.

Amino acid sequence comparisons may be performed using any of thevariety of sequence comparison algorithms and programs known in the art.Such algorithms and programs include, but are by no means limited toBLASTP (Altschul et al. (1997) Nucl. Acids Res. 25: 3389-3402; Schäfferet al. (2001) Nucl. Acids Res. 29: 2994-3005) or BioEdit.

The term “position” refers to a specific amino acid residue present inthe wild-type protein, as identified by a specific numbering of theamino acids. It is apparent to the expert that the insertion or deletionof one or more amino acid residues compared to the wild-type sequenceleads to a different numbering between the wild-type amino acid sequenceand the modified amino acid sequence. For example, if one amino acid isinserted between amino acids 299 and 300 of the wild-type amino acidsequence, the amino acid following the insertion will have the numbering301 in a modified amino acid sequence, while it retains the numbering300 in the wild-type sequence.

The modified 13-hydroperoxyde lyase of the invention may comprise one ormore substitutions selected from the group consisting of V3L, R4K, TSP,S19L, N208H, F340L, Y342F, K352R, K352S, H354Y, F358Y, D359E, V360I,K371P, V372L, T375R, P377S, E382D, P383A, N387K, S388A, D389E, V392M,Q393G, N394E, D399S, D399N and N457K.

The modified 13-hydroperoxide lyase of the present invention maycomprise at least 3 of said substitutions, preferably at least 4, 5, 6,7 or 8, more preferably at least 9, 10, 11, 12, 13 or 14, even morepreferably at least 15, 16, 17 or 18 and most preferably 19, 20, 21, 22,23, 24 or 25 substitutions with respect to the amino acid sequence ofthe wild-type protein according to SEQ ID No. 1.

Preferably, the modified 13-hydroperoxide lyase of the present inventionmay comprise at least the following substitutions: V3L, R4K, T5P andN208H, as compared to the wild-type protein. All the variants disclosedin the present application and showing an enhanced activity have thesesubstitutions. For example, the variant D10A, which is disclosed in thepresent application, comprises these four substitutions, and theadditional substitutions V360I and D399N, as compared to the wild-typeprotein.

The hydroperoxyde lyase of the invention may also comprise the insertionof amino acids K and G between positions 394 and 395 of the amino acidsequence of the wild-type protein. This insertion has the effect thatthe amino acid sequences modified by this insertion are two amino acidslonger than amino acid sequences which do not comprise this insertion.Furthermore, any additional mutations following the insertion will havea different numbering in the wild-type and the modified amino acidsequence. For example, amino acid residue 399 of the wild-type sequencecorresponds to amino acid residue 401 in the modified sequence due tothe insertion.

The modified 13-hydroperoxide lyase of the present invention may furthercomprise the following substitutions: K371P, V372L, T375R, P377S, E382D,P383A, N387K, S388A, D389E, V392M, Q393G, N394E, D399S.

Further examples of the 13-hydroperoxide lyase of the invention,designated GC7, E8B, B7A, C2A, AC5, 4E10 and 9D3, have differentcombinations of amino acid alterations as compared to the amino acidsequence of the wild-type protein. The variant GC7 has 21 amino acidsubstitutions, the variant E8B has 19 amino acid substitutions, thevariant AC5 has 24 amino acid substitutions, the variant C2A has 22amino acid substitutions, the variant B7A has 21 amino acidsubstitutions, the variant 4E10 has 15 amino acid substitutions and 9D3has 25 amino acid substitutions as compared to the amino acid sequenceof the wild-type protein according to SEQ ID No. 1. These seven variantshave the substitutions V3L, R4K, T5P and N208H, as well as thesubstitutions K371P, V372L, T375R, P377S, E382D, P383A, N387K, S388A,D389E, V392M, Q393G, N394E and D399S, as compared to the amino acidsequence of the wild-type protein according to SEQ ID No. 1. Moreover,these variants all additionally have the insertion of amino acids K andG between positions 394 and 395 of the amino acid sequence of thewild-type protein. All positions given herein refer to the position inthe amino acid sequence of the wild-type protein, unless statedotherwise.

Variant GC7 additionally comprises the amino acid substitutions S19L,F340L, V360I and N457K. Variant E8B additionally comprises the aminoacid substitutions F340L and N457K. Variant AC5 additionally comprisesthe amino acid substitutions S19L, F340L, Y342F, K352R, F358Y, V360I andN457K. Variant C2A additionally comprises the amino acid substitutionsF340L, Y342F, F358Y, D359E and V360I. Variant B7A additionally comprisesthe amino acid substitutions S19L, F358Y, V360I and N457K. Variant 4E10additionally comprises the amino acid substitution V360I. Variant 9D3additionally comprises the amino acid substitutions S19L, F340L, Y342F,K352S, H354Y, F358Y, V360I and N457K.

All the isolated variants exemplified (D10A, GC7, E8B, B7A, C2A, AC5,4E10 and 9D3) additionally include the amino acid sequence ATPSSSSPE(SEQ ID NO:18) on the N-terminal end which results in an increasedactivity as compared to enzymes that do not have this insertion. Thisadditional amino acid sequence is derived from 9-hydroperoxide lyase ofCucumis melo (U.S. Pat. No. 7,037,693) and is located at amino acidpositions 1 to 9 of the sequences according to SEQ ID Nos. 2, 4, 6, 8,10, 12, 14 and 16. The expert is aware that the insertion of the aminoacid sequence leads to a different numbering between the wild-type andthe variant proteins. For example, the amino acid at position 3 of thewild-type protein corresponds to the amino acid at position 12 of thevariant.

Most preferably, the modified 13-hydroperoxide lyase according to thepresent invention has an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 2 (variant GC7), 4 (variant E8B), 6 (variantAC5), 8 (variant C2A), 10 (variant B7A), 12 (variant D10A), 14 (variant4E10) and 16 (variant 9D3).

The modified 13-hydroperoxide lyase enzymes of the present inventionshow an increased enzymatic activity compared to the wild-type13-hydroperoxide lyase according to SEQ ID No. 1. The increase inactivity is at least 10% or 20%, preferably 30% or 40%, also preferablyat least 50% or 80%, especially preferably at least 100% or 200%, alsoespecially preferably an increase at least by a factor of 5, 7 or 9,particularly preferably an increase at least by a factor of 10 or 20,also particularly preferably at least by a factor of 50 or 80, and mostpreferably at least by a factor of 100. The term “improved enzymaticactivity” refers to the capability of the modified enzyme to lead to ahigher yield factor than the wild-type enzyme. By “yield factor” it isunderstood here the ratio between the product concentration obtained andthe concentration of biocatalyst (for example, purified recombinantenzyme or an extract from the host cells or the recombinant cellsexpressing the enzyme) in the reaction medium. The present inventorshave shown that the modified 13-hydroperoxide lyases of the presentinvention lead to a 100% conversion of the substrate13-hydroperoxy-octadecatrienoic acid, while the wild-type13-hydroperoxide lyase from guava is only able to convert about 30% ofthe same substrate even at a high concentration of cells.

The 13-hydroperoxide lyase activity can be determined by incubating thepurified enzyme or extracts from host cells or a complete host organismwith a 13-hydroperoxide of a poly-unsaturated fatty acid such aslinolenic acid under appropriate conditions and analysis of the reactionproducts, e.g. by gas chromatography or HPLC analysis. Details aboutenzyme activity assays and analysis of the reaction products are givenbelow in the examples.

The present invention further relates to isolated nucleic acid moleculescomprising a nucleotide sequence encoding a modified 13-hydroperoxidelyase of the present invention. The expert is aware of the fact that dueto the degeneracy of the genetic code a specific amino acid sequence maybe encoded by different nucleic acid sequences. The invention isintended to comprise all nucleotide sequences which code for the13-hydroperoxide lyases of the present invention.

Preferably, the nucleotide sequence coding for the modified13-hydroperoxide lyase of the present invention is selected from thegroup consisting of SEQ ID Nos. 3, 5, 7, 9, 11, 13, 15 and 17, codingfor the protein variants GC7, E8B, AC5, C2A, B7A, D10A, 4E10 and 9D3,respectively.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules present in the natural repository of nucleic acids. Thenucleic acid molecules of the present invention, such as a nucleic acidmolecule with a nucleotide sequence of Nos. 3, 5, 7, 9, 11, 13, 15 or 17or a part thereof, can be isolated or produced using standard molecularbiological techniques and the sequence information provided herein.

The present invention further relates to recombinant nucleic acidmolecules comprising a nucleotide sequence of the present invention.

In terms of the invention, “transgenic” or “recombinant” means, withregard to e.g. a nucleic acid sequence, an expression cassette (=geneconstruct), or a vector containing the nucleic acid sequence accordingto the invention, or an organism transformed with the respective nucleicacid sequences, expression cassettes, or vectors, all of thoseconstructs being produced by means of genetic technologies, that either

-   a) the nucleic acid sequence according to the invention, or-   b) a genetic control sequence functionally linked to the nucleic    acid sequence according to the invention, such as a promoter, or-   c) a) and b)

is not in its natural genetic environment, or has been modified bygenetic techniques, the modification being, for example, a substitution,addition, deletion, inversion, or insertion of one or several nucleotideresidues. Natural genetic environment means the natural genomic orchromosomal locus in the parental organism, or the existence in agenomic library.

Another aspect of the invention pertains to organisms or host cells intowhich a recombinant expression vector of the invention has beenintroduced. The terms “host cell”, “transgenic cell” and “recombinantcell” are used interchangeably herein. It is understood that such termsrefer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation(s) orenvironmental influences, such progeny may not, in fact, be identical tothe parent cell, but are still included within the scope of the term asused herein.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,plants, insect cells and mammalian cells.

It is obvious to the person skilled in the art that the nucleic acidsequence which encodes a 13-HPOL and which is used for the production ofthe transgenic cell may have to be adjusted to the organism specificcodon usage. The codon usage can be determined with computer analyses ofother known genes of the selected organism.

After their insertion into a cell, the nucleic acids used in the methodcan either be located on a separate plasmid, or advantageously beintegrated into the genome of the host cell. In the case of integrationinto the genome, the integration can occur randomly, or by means of sucha recombination that the native gene is replaced by the inserted copy,which causes the modulation of cellular 13-HPOL expression, or by usinga gene in trans so that the gene is functionally linked to a functionalexpression unit which contains at least one sequence ensuring theexpression of a gene, and at least one sequence ensuring thepolyadenylation of a functionally transcribed gene.

In addition to the nucleic acid sequence coding for the 13-HPOL to betransferred, the recombinant nucleic acid molecules which are used forthe expression of the 13-HPOL further comprise regulatory elements.Which precise regulatory elements these vectors have to contain dependsin each case on the process in which these vectors are to be used. Theperson skilled in the art knows which regulatory and other elements arecombinant nucleic acid molecule has to contain.

Typically, the regulatory elements which are part of the vectors aresuch that allow for the transcription and, if desired, for thetranslation in the bacterial cell, yeast cell, insect cell, mammaliancell or plant cell. Depending on the organism selected, this can mean,for example, that the gene is only expressed and/or overexpressed afterinduction, or that it is expressed and/or overexpressed immediately. Forexample, these regulatory sequences are sequences to which inductors orrepressors bind, thereby regulating the expression of the nucleic acid.In addition to these new regulatory sequences, or instead of thesesequences, the natural regulation of the sequences upstream of theactual structural genes may still be existent, and may possibly havebeen genetically modified so that the natural regulation is disabled,and the expression of the genes is increased. The recombinant nucleicacid molecule, however, can also be constructed in a simpler manner,i.e. no additional regulation signals are inserted upstream of thenucleic acid sequence, and the natural promoter with its regulation hasnot been removed. Instead, the natural regulatory sequence has beenmutated in such a manner that regulation no longer occurs, and/or geneexpression is increased. To increase the activity, these alteredpromoters can also be inserted singly, in the form of partial sequences,upstream of the natural gene. Furthermore, the gene construct can alsoadvantageously contain one or more so-called enhancer sequences whichare functionally linked to the promoter, and which allow an increasedexpression of the nucleic acid sequence. Additional useful sequences,such as additional regulatory elements or terminators, can also beinserted at the 3′ end of the DNA sequences.

In principle, it is possible to use any of the natural promoters withtheir regulation sequences for the method according to the invention.However, it is also possible and advantageous to use synthetic promotersonly or in addition.

The vectors according to the invention, as the regulatory elements, canadditionally comprise, e.g. enhancer elements. They may also containresistance genes, replication signals, and additional DNA regions, whichenable propagation of the vectors in bacteria, such as E. coli. Theregulatory elements also comprise sequences which effect a stabilizationof the vectors in the host cells. Such regulatory elements particularlycomprise sequences facilitating stable integration of the vector intothe host genome, or an autonomous replication of the vector in thecells. Such regulatory elements are known to the person skilled in theart.

The so-called termination sequences are sequences which ensure theproper termination of transcription or translation. If the transferrednucleic acids are to be translated, the termination sequences aretypically stop codons and respective regulatory sequences; if thetransferred nucleic acids are only to be transcribed in eukaryotes, theyare generally poly(A) sequences.

As used herein, the term “vector” relates to a recombinant nucleic acidmolecule which can transport another nucleic acid, to which it is bound,into a cell. A vector type is a “plasmid” representing a circular doublestranded DNA loop, into which additional DNA segments can be ligated.Another vector type is a viral vector, wherein additional DNA segmentscan be ligated into the viral genome. Certain vectors can replicateautonomously in a host cell into which they have been inserted (e.g.bacterial vectors with a bacterial replication origin). Other vectorsare advantageously integrated into the genome of a host cell wheninserted in the host cell, and thereby replicated together with the hostgenome. Also, certain vectors can control the expression of genes towhich they are functionally linked. These vectors are called here“expression vectors.”. Usually, expression vectors suitable for DNArecombination techniques are of the plasmid type. In the presentdescription “plasmid” and “vector” can be used interchangeably, sincethe plasmid is the vector type most often used. However, the inventionis also intended to comprise other types of expression vectors, such asviral vectors which fulfil similar functions. Furthermore, the term“vector” is also intended to comprise other vectors known to the personskilled in the art, such as phages, viruses, such as SV40, CMV, TMV,transposons, IS elements, phasmids, phagemids, cosmids, linear orcircular DNA.

In a recombinant expression vector, the term “operatively linkedthereto” or “functionally linked thereto” means that the nucleotidesequence of interest is linked to the regulatory sequence(s) in such away that the expression of the nucleotide sequence is possible, and thatboth sequences are linked to each other in such a way so as to fulfilthe predicted function ascribed to the sequence.

The term “regulatory sequence” is intended to comprise promoters,enhancers, and other expression control elements (e.g. polyadenylationsignals). These regulation sequences are described e.g. in Goeddel: GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), or in Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, CRC Press, Boca Raton, Fla.,publisher: Glick and Thompson, Chapter 7, 89-108. Regulatory sequencescomprise those sequences which regulate the constitutive expression of anucleotide sequence in many types of host cells, and those sequenceswhich regulate the direct expression of the nucleotide sequence only incertain host cells under certain conditions. The person skilled in theart knows that the design of the expression vector can depend onfactors, such as the choice of the host cell to be transformed, thedesired extent of the protein expression, etc.

The recombinant expression vectors used for the expression of the13-HPOL can be active in both prokaryotic and eukaryotic cells. This isadvantageous, since intermediate steps of the vector construction areoften performed in microorganisms for the sake of simplicity. Thesecloning vectors contain a replication signal for the respectivemicroorganism, and a marker gene for the selection of successfullytransformed bacterial cells. Suitable vectors for expression inprokaryotic organisms are known to the person skilled in the art; theyinclude e.g. E. coli pEXP5-NT/TOPO, pMAL series, pLG338, pACYC184, thepBR series, such as pBR322, the pUC series, such as pUC18, or pUC19, theM113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, λgt11, or pBdC1, Streptomyces pIJ101, pIJ364,0.1702, or pIJ361, Bacillus pUB110, pC194, or pBD214, CorynebacteriumpSA77, or pAJ667.

In another embodiment the expression vector represents a yeastexpression vector or a baculovirus expression vector.

The above named vectors provide only a small overview of possiblesuitable vectors. Additional plasmids are known to the person skilled inthe art and are described in e.g.: Cloning Vectors (publisher Pouwels,P. H. et al. Elsevier, Amsterdam, N.Y.-Oxford, 1985). For additionalsuitable expression systems for prokaryotic and eukaryotic cells seechapters 15 and 16 of Sambrook and Russell, vide supra.

In another embodiment of the method, the 13-HPOL can be expressed insingle-celled plant cells (such as algae), see Falciatore et al. (1999)Marine Biotechnology 1(3): 239-251 and the literature cited therein, andin plant cells of higher plants (e.g. spermatophytes, such as cropplants). Examples for plant expression vectors comprise thoseextensively described in: Becker et al. (1992) Plant Mol. Biol. 20:1195-1197; and Bevan (1984) Nucl. Acids Res. 12: 8711-8721; Vectors forGene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1,Engineering and Utilization, publisher: Kung and R. Wu, Academic Press,1993, S. 15-38.

The gene to be expressed must, as described above, be functionallylinked to a suitable promoter which regulates the gene expression in atime specific, cell specific or tissue specific manner.

In order to insert the 13-HPOL nucleotide sequence into the expressionvectors, they are advantageously subjected to an amplification andligation in the known manner. Preferably, one proceeds in accordancewith the protocol of the Pfu DNA polymerase, or a Pfu/Taq DNA polymerasemixture. The primers are selected in accordance with the sequence to beamplified. Advantageously, the primer should be selected so that theamplified DNA comprises the entire codogenic sequence from the startcodon to the stop codon. Advantageously, the amplified DNA is analyzedsubsequent to the amplification. For example, the analysis can be madein respect of quality and quantity after gel electrophoretic separation.The amplified DNA can then be purified according to a standard protocol(e.g. Qiagen). An aliquot of the purified amplified DNA is thenavailable for the subsequent cloning.

Suitable cloning vectors are generally known to the person skilled inthe art. These particularly include vectors which are replicable inmicrobial systems, i.e. especially vectors which allow for an efficientcloning and expression in bacteria, yeasts or fungi, and/or which allowfor the stable transformation of plants. Especially worth mentioning arevarious binary and co-integrated vector systems suitable for the T-DNAmediated transformation of plants. These binary vectors include vectorsof the series pBIB-HYG, pPZP, pBecks, pGreen. According to theinvention, Bin19, pBI101, pBinAR, pGPTV and pCAMBIA are preferred. Anoverview of binary vectors and their use is provided by Hellens et al.(2000) Trends in Plant Science 5: 446-451.

For the preparation of the vector, the vectors can initially belinearized by means of restriction endonuclease(s) and thenenzymatically modified in any suitable way. The vector is then purifiedand an aliquot is used for cloning. During cloning the enzymatically cutand if necessary purified amplified DNA is linked to similarly preparedvector fragments by means of a ligase. A certain nucleic acid construct,or vector construct, or plasmid construct, may have one, or evenseveral, codogenic gene regions. Preferably, the codogenic gene regionsin these constructs are functionally linked to regulatory sequences asdescribed above. The constructs can be advantageously cultivated inmicroorganisms, especially in E. coli and Agrobacterium tumefaciens, ina suitable medium, and stably propagated under selection conditions. Thecells are then harvested and lysed and the plasmid is extractedtherefrom. This allows a transfer of heterologous DNA intomicroorganisms or plants.

With the advantageous use of cloning vectors, the nucleic acids and thenucleic acid constructs according to the invention can be inserted intoorganisms, such as microorganisms, or plants, and used for planttransformation. The nucleic acids used in the method, the nucleic acidsand nucleic acid constructs, and/or vectors according to the invention,can therefore be used for the genetic modification of a broad spectrumof organisms.

In terms of the invention, the term “transgenic cell” means, asdescribed above, that the nucleic acids used in the method are not attheir natural site in the genome of the cell, whereby the nucleic acidscan expressed in homologous or heterologous host cells. However,transgenic also means that although the nucleic acids according to theinvention are located at their natural site in the genome of anorganism, i.e. the site of the 13-HPOL, the sequence has been changedcompared to the natural sequence, and/or the regulatory sequences of thenatural sequences have been modified. Preferably, transgenic is to beunderstood as the expression of the nucleic acids according to theinvention at a non-natural site in the genome, i.e. the nucleic acidsare homologously, or preferably heterologously expressed.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,plants, insect cells and mammalian cells.

Preferred host organisms according to the present invention are bacteriasuch as the ones of the genus Escherichia, fungi such as for exampleMortierella, Saprolegnia or Pythium, yeasts such as Saccharomyces,cyanobacteria, ciliates, algae or protozoa such as dinoflagellates suchas Crypthecodinium. Industrially used suitable microorganisms include,but are not limited to Gram negative bacteria such as E. coli, Grampositive bacteria such B. subtilis, fungi such as A. niger, A. nidulans,N. crassa; yeasts such as S. cerevisiae, K. lactis, H. polymorpha, P.pastoris, Y. lipolytica; actinomycetes such as Streptomyces sp. Severalof these microorganisms are described in the ‘Manual of IndustrialMicrobiology and Biotechnology’ (editors in chief: A. L. Demain, J. E.Davies; ASM Press).

Further suitable host cells can be derived from: Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Suitable expression strains, e.g. with a lowerprotease activity are described in: Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128.

Most preferably, E. coli cells are used. Suitable E. coli strains are,for example, MC1061, BL21, JM101, JM105, JM109 and DH5α.

According to the invention, the term “transgenic plant” comprises theplant in its entirety, as well as all parts of the plant in which theexpression of the 13-HPOL proteins according to the invention isincreased. This includes all parts of the plant and plant organs, suchas leaf, stem, seed, root, tubers, anthers, fibers, root hair, stalk,embryos, calli, cotelydons, petioles, crop material, plant tissue,reproductive tissue, cell cultures derived from the transgenic plant,and/or which can be used to produce the transgenic plant.

The plants used for the method according to the invention can inprinciple be any plant which is to be made resistant to a pathogeninfestation. Preferably, it is a monocotyledonous or dicotyledonous suchas an agricultural plant, a food plant or a fodder plant.

Suitable mammalian cells include for example NIH3T3 cells, CHO cells,COS cells, 293 cells, Jurkat cells, BHK cells and HeLa cells.

The specific expression of the 13-HPOL protein in the transgenic cellsaccording to the invention can be proven and tracked by means of commonmolecular biological and biochemical methods. The person skilled in theart knows these techniques and is easily able to select suitabledetection methods, such as a Northern Blot analysis for the detection of13-HPOL-specific RNA, or for the determination of the amount ofaccumulation of 13-HPOL-specific RNA, or a Southern Blot, or PCR,analysis for the detection of DNA sequences encoding the 13-HPOL. Theprobe or primer sequences used for this purpose can either be identicalto the sequences given in SEQ ID Nos. 3, 5, 7, 9 11, 13, 15 or 17, orshow some slight differences to these sequences.

If microorganisms are used in the present invention, these organisms arepreferably grown under standard conditions in a fermentation process ina manner known to the expert.

The term “standard conditions” refers to the cultivation of amicroorganism in a standard medium. The temperature, pH and incubationtime can vary as described below.

The temperature should be usually in a range between 15° C. and 45° C.,but the range may be higher, up to 105° C. for thermophilic organisms.The temperature can be kept constant or can be altered during theexperiment.

The pH of the medium may be in the range of 5 to 8.5, preferably around7.0, and can be maintained by the addition of buffers to the media. Anexemplary buffer for this purpose is a potassium phosphate buffer.Synthetic buffers such as MOPS, HEPES, ACES and others can alternativelyor simultaneously be used. It is also possible to maintain a constantculture pH through the addition of an acid or base, such as acetic acid,sulfuric acid, phosphoric acid, NaOH, KOH or NH₄OH during growth. Ifcomplex medium components such as yeast extract are utilized, thenecessity for additional buffers may be reduced, due to the fact thatmany complex compounds have high buffer capacities. If a fermentor isutilized for culturing the microorganisms, the pH can also be controlledusing gaseous ammonia.

The incubation time is usually in a range from several hours to severaldays. This time is selected in order to permit the maximal amount ofproduct to accumulate in the broth.

The disclosed growth experiments can be carried out in a variety ofvessels, such as microtiter plates, glass tubes, glass flasks or glassor metal fermentors of different sizes. For screening a large number ofclones, the microorganisms should be cultured in microtiter plates,glass tubes or shake flasks, either with or without baffles. Preferably100 ml or 250 shake flasks are used, filled with about 10% (by volume)of the required growth medium. The flasks should be shaken on a rotaryshaker (amplitude about 25 mm) using a speed-range of about 100-300 rpm.Evaporation losses can be diminished by the maintenance of a humidatmosphere; alternatively, a mathematical correction for evaporationlosses should be performed.

If genetically modified clones are tested, an unmodified control cloneor a control clone containing the basic plasmid without any insertshould also be tested.

The standard culture conditions for each microorganism used can be takenfrom the textbooks, such as Sambrook and Russell, Molecular Cloning—Alaboratory manual, Cold Spring Harbour Laboratory Press, 3^(rd) edition(2001).

E.g., E. coli and C. glutamicum strains are routinely grown in MB or LBand BHI broth (Follettie, M. T. et al. (1993) J. Bacteriol. 175:4096-4103, Difco Becton Dickinson). Usual standard minimal media for E.coli are M9 and modified MCGC (Yoshihama et al. (1985) J. Bacteriol.162: 591-507; Liebl et al. (1989) Appl. Microbiol. Biotechnol. 32:205-210.). Other suitable standard media for the cultivation of bacteriainclude NZCYM, SOB, TB, CG12 ½ and YT.

“Standard media” within the meaning of the present invention areintended to include all media which are suitable for the cultivation ofthe microorganisms of the present invention and include both enrichedand minimal media.

“Minimal media” are media that contain only the minimal necessities forthe growth of wild-type cells, i.e. inorganic salts, a carbon source andwater.

In contrast, “enriched media” are designed to fulfil all growthrequirements of a specific microorganism, i.e. in addition to thecontents of the minimal media they contain for example growth factors.

Antibiotics may be added to the standard media in the following amounts(micrograms per milliliter): ampicillin, 50; kanamycin, 25; nalidixicacid, 25 to allow for the selection of transformed strains.

Suitable media consist of one or more carbon sources, nitrogen sources,inorganic salts, vitamins and trace elements. Preferred carbon sourcesare sugars, such as mono-, di-, or polysaccharides. For example,glucose, fructose, mannose, galactose, ribose, sorbose, lactose,maltose, sucrose, raffinose, starch or cellulose may serve as very goodcarbon sources.

It is also possible to supply sugar to the media via complex compoundssuch as molasses or other by-products from sugar refinement. It can alsobe advantageous to supply mixtures of different carbon sources. Otherpossible carbon sources are alcohols and organic acids, such asmethanol, ethanol, acetic acid or lactic acid. Nitrogen sources areusually organic or inorganic nitrogen compounds, or materials whichcontain these compounds. Exemplary nitrogen sources include ammonia gasor ammonia salts, such as NH₄Cl or (NH₄)₂SO₄, NH₄OH, nitrates, urea,amino acids or complex nitrogen sources like corn steep liquor, soy beanflour, soy bean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include thechloride, phosphate or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating compounds can be added to the medium to keep the metal ions insolution. Particularly useful chelating compounds includedihydroxyphenols, like catechol or protocatechuate, or organic acids,such as citric acid. It is typical for the media to also contain othergrowth factors, such as vitamins or growth promoters, examples of whichinclude biotin, riboflavin, thiamin, folic acid, nicotinic acid,pantothenate and pyridoxin. Growth factors and salts frequentlyoriginate from complex media components such as yeast extract, molasses,corn steep liquor and others. The exact composition of the mediacompounds depends strongly on the immediate experiment and isindividually decided for each specific case. Information about mediaoptimization is available in the textbook “Applied Microbiol.Physiology, A Practical Approach (eds. P. M. Rhodes, P. F. Stanbury, IRLPress (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible toselect growth media from commercial suppliers, like standard 1 (Merck)or BHI (brain heart infusion, DIFCO) or others.

Moreover, it may also be advantageous to add the heme precursordelta-aminolevulinic acid to the medium in order to improve theexpression efficiency of active recombinant lyase.

All medium components should be sterilized, either by heat (20 minutesat 1.5 bar and 121° C.) or by sterile filtration. The components caneither be sterilized together or, if necessary, separately.

The preparation of standard media used for the cultivation of bacteriausually does not involve the addition of single amino acids. Instead, inenriched media for use under standard culture conditions a mixture ofamino acids such as peptone or trypton is added.

The transgenic cells according to the present invention can becultivated in any suitable manner, for example by batch cultivation orfed-batch cultivation.

“Batch cultivation” within the meaning of the present invention is acultivation method in which culture medium is neither added norwithdrawn during the cultivation.

A “fed-batch method” within the meaning of the present invention is acultivation method in which culture medium is added during thecultivation, but no culture medium is withdrawn.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins.

Fusion vectors add a number of amino acids to a protein encoded by theinserted nucleic acid sequence, usually to the amino terminus of therecombinant protein but also to the C-terminus or fused within suitableregions in the proteins. Such fusion vectors typically serve threepurposes:

-   1) to increase expression of recombinant protein;-   2) to increase the solubility of the recombinant protein; and-   3) to aid in the purification of the recombinant protein by    providing a ligand for affinity purification.

Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase.

Typical fusion expression vectors include pQE (Qiagen), pGEX (PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione 5-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the recombinant protein.

Examples for C. glutamicum vectors can be found in the Handbook ofCorynebacterium 2005 Eggeling, L. Bott, M., eds., CRC press USA.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69: 301-315), pLG338, pACYC184,pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, egtll, pBdC1, and pET lld (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89; Pouwels et al., eds. (1985) Cloning Vectors.Elsevier: New York ISBN 0 444 904018). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET lld vectorrelies on transcription from a T7 gnlO-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gnl). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174 (DE3) from a resident Xprophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter. For transformation of other varieties of bacteria,appropriate vectors may be selected. For example, the plasmids pIJ101,pIJ364, pIJ702 and pIJ361 are known to be useful in transformingStreptomyces, while plasmids pUB110, pC194, or pBD214 are suitable fortransformation of Bacillus species. Several plasmids of use in thetransfer of genetic information into Corynebacterium include pHM1519,pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors.Elsevier: New York IBSN 0 444 904018).

In another embodiment, the protein expression vector is a yeastexpression vector. Examples of vectors for expression in yeast (S.cerevisiae) include pYepSecl (Baldari, et al. (1987) Embo J. 6:229-234), 2i, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz(1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectorsand methods for the construction of vectors appropriate for use in otherfungi, such as the filamentous fungi, include those detailed in: van denHondel, C. A. M. J. J. & Punt, P. J. (1991) in: Applied MolecularGenetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, CambridgeUniversity Press: Cambridge, and Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York (ISBN 0 444 904018).

Vector DNA can be introduced into prokaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection”, “conjugation” and “transduction”are intended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e. g., linear DNA or RNA (e. g., alinearized vector or a gene construct alone without a vector) or nucleicacid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid,transposon or other DNA into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemical-mediated transfer, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2003), and other laboratorymanuals.

In order to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as, but not limited to, G418, hygromycin, kanamycin,tetracycline, neomycineampicillin (and other pencillins),cephalosporins, fluoroquinones, naladixic acid, chloramphenicol,spectinomycin, erythromycin, streptomycin and methotrexate.

Other selectable markers include wild type genes that can complementmutated versions of the equivalent gene in a host or starting strain.For example, an essential gene for growth on a minimal medium, such asserA, can be mutated or deleted from the genome of a C. glutamicumstarting or host strain of the invention as described herein above tocreate a serine auxotroph. Then, a vector containing a wild type orother functional copy of a serA gene can be used to select fortransformants or integrants. Nucleic acid encoding a selectable markercan be introduced into a host cell on the same vector as that encodingthe above-mentioned modified nucleic acid sequences or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e. g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

When plasmids without an origin of replication and two different markergenes are used, it is also possible to generate marker-free strainswhich have part of the insert inserted into the genome. This is achievedby two consecutive events of homologous recombination (see also Beckeret al. (2005) Appl. Environ. Microbiol. 71 (12): 8587-8596).

In another embodiment, recombinant microorganisms can be produced whichcontain selection systems which allow for regulated expression of theintroduced gene. For example, inclusion of one of the above-mentionednucleic acid sequences on a vector placing it under control of the lacoperon permits expression of the gene only in the presence of IPTG. Suchregulatory systems are well known in the art.

There are plurality of known techniques available for inserting DNA intoa plant host cell, and the person skilled in the art will have nodifficulty in finding the most suitable method in each case. Thesetechniques comprise the transformation of plant cells with T-DNA usingAgrobacterium tumefaciens or Agrobacterium rhizogenes as transformationagent, the fusion of protoplasts, the direct gene transfer of isolatedDNA into protoplasts, the electroporation of DNA, the insertion of DNAby means of the biolistic method, as well as other possibilities. Bothstable and transient transformants can be generated in this manner.

Of course, the plant cells containing the nucleic acid moleculesaccording to the invention may also be further cultivated in the form ofa cell culture (including protoplasts, calli, suspension cultures, andthe like).

Besides using transgenic organisms or cells such as bacterial, yeast,insect, mammalian or plant cells and fungi or algae, the 13-HPOL enzymesof the present invention may also be expressed in a cell-free systemusing a suitable expression vector. The cell free expression is acoupled transcription and translation reaction to produce activerecombinant protein in high amounts in vitro and can be obtainedcommercially.

The present invention further relates to a method for preparing amodified 13-hydroperoxide lyase polypeptide of the present invention byculturing one or more transgenic cells according to the presentinvention under conditions which permit expression of the polypeptideand optionally recovering the polypeptide.

The term “culturing of cells” means that the cells are grown underconditions that allow proliferation of the cells and expression of thepolypeptide. These conditions include the medium containing componentswhich are necessary for the metabolism of the cells, the temperature,the pH and the incubation time, as discussed above.

The polypeptide may be recovered by any purification method which isknown in the art, e.g. gel filtration or chromatographic methods.Preferably, the protein is expressed as a fusion protein together with anumber of amino acids which provide a ligand for affinity purification,such as glutathione S-transferase, maltose binding protein or protein A.As discussed above, these ligands may be removed after purification bytreatment of the fusion protein with a protease.

The present invention further relates to the use of the modified13-hydroperoxide lyase of the present invention for cleaving a13-hydroperoxide of a polyunsaturated fatty acid into an aldehyde and anoxocarboxylic acid.

The term “polyunsaturated fatty acid” refers to fatty acids with atleast two double bonds.

“Fatty acid hydroperoxides” are formed by oxidation of unsaturated fattyacids. In particular, 13-hydroperoxides are formed by the oxidation ofunsaturated fatty acids at carbon atom 13. If linoleic acid or linolenicacid having 18 carbon atoms is used as the polyunsaturated fatty acid inthe method of the present invention, the action of the 13-hydroperoxidelyase of the present invention leads to the production of a C₆-aldehydeand a C12-oxocarboxylic acid. For example, n-hexanal is produced fromthe 13-hydroperoxide of linoleic acid and 3-(Z)-hexen-1-al is producedfrom the 13-hydroperoxide of alpha-linolenic acid.

More particularly the reaction product obtained by cleavage of13-hydroperoxydes of linseed oil hydrolysate (mixture of13-hydroperoxydes of linoleic acid and of linolenic acid) by the 13hydroperoxyde lyase of the invention is a mixture of n-hexanal,3-(Z)-hexen-1-al and 2-(E)-hexen-1-al.

The C₆-aldehydes produced can then be used as flavour and/or fragranceingredients or further reacted to yield other products such as thecorresponding alcohols. The desired products obtained can be separatedfrom the reaction medium by means of steam distillation and/orextraction with an inert organic solvent.

Therefore the present invention relates to a method of producing analdehyde, comprising the steps of:

-   a) contacting a 13-hydroperoxide of a polyunsaturated fatty acid    with the modified 13-hydroperoxide lyase of the present invention;    and-   b) recovering the produced aldehyde.

Preferably said method further comprises, prior to step a), the step ofproviding a modified 13-hydroperoxide lyase of the present invention.

Preferably the present invention relates to a method of producing amixture of n-hexanal, 3-(Z)-hexen-1-al and 2-(E)-hexen-1-al, comprisingthe steps of

-   a) contacting a 13-hydroperoxyde of linseed oil hydrolysate with the    modified 13-hydroperoxyde lyase of the invention; and-   b) recovering the mixture of hexanal, 3-(Z)-hexen-1-al and    2-(E)-hexen-1-al produced in step a).

More preferably said method further comprises, prior to step a), thestep of providing a modified 13-hydroperoxide lyase of the presentinvention.

The 13-hydroperoxide lyase of the present invention may be provided indifferent ways.

First, recombinant microbial cells (e.g. E. coli) may be lysed, e.g. bysonification, osmotic shock, freeze-thaw cycles or by the use of celllysis kits (e.g. CelLytic™ of Sigma) and then the lysate obtained may bedirectly added to a solution of fatty acid hydroperoxides to form thealdehyde and the oxocarboxylic acid.

Second, the hydroperoxides may be produced in vivo in a transgenic cellor a transgenic organism such as microbial cells, plants, plant cellcultures, fungi, mammalian cells, insect cells and cyanobacteria.

Third, the 13-HPOL may be purified from the transgenic organism.Preferably, the nucleotide sequence coding for the 13-HPOL is clonedinto a vector which contains a nucleotide sequence coding for anaffinity tag such as cellulose binding protein, maltose binding protein,glutathione-S-transferase or a His-tag. After transcription andtranslation of the DNA a fusion protein is formed which can then beeasily purified by means of affinity chromatography. Vectors and methodswhich are useful for the production and purification of the fusionprotein are well-known to those skilled in the art. After thepurification, the affinity tag may be removed by the action of aprotease such as Factor X. However, such affinity tags provide increasedactivity of the recombinant fusion protein due to improved proteinfolding/solubility etc. These and other tags also allow theimmobilisation of the fusion protein on cheap matrices such as amylose,cellulose, which can be used in reactors for the stabilization andrecycling of the recombinant enzyme.

The term “contacting” means that the enzyme 13-hydroperoxide lyase andits substrate, the 13-hydroperoxide of a polyunsaturated fatty acid, arebrought together under suitable reaction conditions to enable thereaction. The expert knows which reaction conditions have to be selectedfor the reaction to occur. Further, examples for such reactionconditions are given in the examples below.

The produced aldehyde can be recovered by any methods known to theexpert, for example by means of steam distillation and/or extractionwith an inert organic solvent. The reaction products can then beanalyzed by means of gas chromatography or HPLC.

The substrate for the 13-hydroperoxide lyase, i.e. the 13-hydroperoxideof a polyunsaturated fatty acid, may be produced by the activity of a13-lipoxygenase. Oxygenases catalyze the regio- and stereo specificdioxygenation of polyunsaturated fatty acids containing at least one1-(Z), 4-(Z)-pentadiene system, e.g. linoleic acid, linolenic acid andarachidonic acid. 13-lipoxygenase has a specificity for the C₁₃-positionwithin the fatty acid, i.e. it oxygenates preferably at carbon atom 13of the hydrocarbon backbone of the fatty acid leading to13-hydroperoxides of the fatty acid.

The 13-lipoxygenase may be provided in the form of homogenized soy beanswhich are the cheapest source of 13-lipoxygenase (see e.g. U.S. Pat. No.5,464,761; U.S. Pat. No. 6,780,621). Another way to provide the13-lipoxygenase is recombinant expression. The amino acid and nucleotidesequence of a prokaryotic lipoxygenase with C₁₃-specificity is disclosedin EP 06123710.3 and in Lang and Feussner (2007) Phytochemistry 68(8):1120-1127 as well as in Koeduka et al. (2007) Curr. Microbiol. 54(4):315-319. The recombinant 13-lipoxygenase may be produced in the samecell as the 13-hydroperoxide lyase or it may be produced in a separatecell. If it is produced within the same cell, the correspondingaldehydes may be produced from the hydroperoxides immediately after thelatter are formed. The 13-lipoxygenase can be used in the form of a celllysate, a cell suspension or of a purified protein as described abovefor the 13-hydroperoxide lyase.

The aldehydes produced by the method of the present invention may bereduced to their corresponding alcohols by various means, e.g. by addingfresh baker's yeast to the reaction solution. The yeast contains anactive alcohol dehydrogenase enzyme that reduces the aldehyde.Alternatively, isolated alcohol dehydrogenases or chemical agents suchas KBH₄ or NaBH₄ may be used for reducing the aldehyde to thecorresponding alcohol.

The isolation of the variant 13-hydroperoxide lyases of the presentinvention and their characterization is described below in the examples.The following examples should not be construed as limiting. The contentof all literature, patent applications, patents and published patentapplications cited in this patent application is incorporated herein byreference.

EXAMPLES Example 1

Molecular Evolution of Wild-Type Guava 13-hydroperoxide Lyase

The modified 13-hydroperoxide lyases of the present invention wereobtained by several rounds of mutagenesis and shuffling of the DNAcoding for the guava 13-hydroperoxide lyase according to SEQ ID No. 1and additional DNA sequences coding for a 9-hydroperoxide lyase from C.melo and DNA sequences coding for 13-hydroperoxide lyases from C.sinensis and N. attenuata. The mutagenesis steps were performed bysubjecting the DNA to the EvoSight™ method as described in WO2006/003298. DNA shuffling steps were performed by subjecting the DNA tothe L-Shuffling™ method as described in WO 00/09679. DNA coding for theguava 13-hydroperoxide lyase according to SEQ ID No. 1 was fragmented,mixed with fragmented additional DNA coding for a 9 or 13-hydroperoxidelyase from C. melo, C. sinensis and N. attenuata and reassembled throughrecursive steps of denaturation/hybridization on a matrix/ligation.High-throughput screening of the mutant proteins at each round ofmolecular evolution was done by monitoring the substrate consumption at234 nm Identified improved variants were validated by chromatographicanalysis of both the substrate and the products.

Example 2

Cloning of the Modified 13-hydroperoxide Lyases into AppropriateExpression Vectors

The nucleotide sequences coding for the modified 13-hydroperoxy lyasesGC7, E8B, AC5, C2A, B7A, D10A, E8B and 9D3 as depicted in SEQ ID Nos. 3,5, 7, 9, 11, 13, 15 and 17, respectively, were cloned into the vectorpMAL-c2X (New England Biolabs) by using a 5′ linker containing an EcoRIsite and a 3′ linker containing a BamHI site. The list of the differentplasmids obtained is shown in Table 1.

TABLE 1 Examples of recombinant plasmids Plasmid Description pB7#AEcoRI - BamHI fragment B7#A in pMAL-c2X pAC#5EcoRI - BamHI fragment AC#5 in pMAL-c2X pC2#AEcoRI - BamHI fragment C2#A in pMAL-c2X pE8#BEcoRI - BamHI fragment E8#B in pMAL-c2X pGC#7EcoRI - BamHI fragment GC#7 in pMAL-c2X pD10#AEcoRI - BamHI fragment D10#A in pMAL-c2X p4E#10EcoRI - BamHI fragment 4E#10 in pMAL-c2X p9D#3EcoRI - BamHI fragment 9D#3 in pMAL-c2X pWTEcoRI - BamHI DNA fragment coding for the protein of SEQ ID NO. 1 withan N-terminal insertion comprising the amino acids ATPSSSSPE(SEQ ID NO: 18)

Example 3

Fatty Acid Hydroperoxide Cleavage by the Modified 13-hydroperoxideLyases as Compared to the ‘Wild-Type’ Enzyme

Experiment 1

E. coli MC1061 cells transformed with the plasmids pB7#A, pAC#5, pC2#A,pE8#B, pGC#7, p4E#10, p9D#3, pD10#A and pWT of Table 1 were cultivatedin shake flask cultures in 25 ml of LB medium with 100 ppm of ampicillinat 180 rpm and at 20° C. for 30 hours without induction. Cultures werecentrifuged and the pellet re-suspended in 100 mM phosphate buffer pH7.6 and adjusted to an optical density of OD₆₀₀=10 for all transformedcells except those transformed with the plasmid pD10#A, for which theoptical density was adjusted to OD₆₀₀=25. The catalytic activity of the13-HPOL enzyme variants was determined as follows: 100 μl (10% v/v) ofcell suspension (0D₆₀₀=10, respectively OD₆₀₀=25) were added to 900 μlof a solution containing 84.3 g kg⁻¹ of fatty acid hydroperoxides(including about 75% of the substrate 13-HPOT) previously produced withlinseed oil hydrolysate as a source of fatty acids and ground soybeansas the source of the 13-lipoxygenase. 20 mg of horse liver alcoholdehydrogenase as reducing agent and 330 mg of NADH were added understirring. The reaction was carried out at room temperature for 2minutes. An aliquot of 100 μl of the reaction mixture was diluted with900 μl of water, extracted with one volume of ethyl acetate and assayedby gas chromatography for the presence of 3-(Z)-hexen-1-ol. Theconsumption of the fatty acid hydroperoxides was followed by HPLC. Themolar yield is expressed as the ratio between the amount of3-(Z)-hexen-1-ol obtained during the reaction and the amount of3-(Z)-hexen-1-ol one could theoretically obtain from the amount of13-HPOT present in the reaction.

The results of this experiment are listed in Table 2, showing theimproved enzymatic activity of the modified lyases compared to therecombinant wild-type enzyme (pWT).

TABLE 2 Activity of modified hydroperoxide lyases Soluble alcoholdehydrogenase from horse liver and NADH was used as the reducing agent.3-(Z)-hexen-1-ol Biocatalyst Molar 13HPOT concentration yield conversionPlasmid (OD₆₀₀) (g l⁻¹) (%) (%) pB7#A 1 4.6 31 38 pAC#5 1 4.0 27 38pC2#A 1 5.3 35 43 pE8#B 1 4.8 32 44 pGC#7 1 4.3 29 46 p4E#10 1 3.9 26 40p9D#3 1 3.8 25 38 pD10#A 2.5 2.8 18.7 58 pWT 1 0.2 1.3 <10*  *within theanalytical error margin

Experiment 2

E. coli MC1061 cells harboring the plasmids pB7#A, pAC#5, pC2#A, pE8#B,pGC#7, p4E#10, pD10#A, p9D3 and pWT of Table 1 were cultivated in shakeflask cultures in 25 ml of LB medium containing 100 ppm of ampicillin at180 rpm and at 20° C. for 30 hours without induction. Cultures werecentrifuged and the pellet re-suspended in 100 mM phosphate buffer pH7.6. The cell density was adjusted to an optical density of OD₆₀₀=20 forall transformed cells except those transformed with the plasmid pD10#A,for which the optical density was adjusted to OD₆₀₀=10. The catalyticactivity was determined as follows: 100 μl (10% v/v) of cell suspension(OD₆₀₀=20, respectively OD₆₀₀=10) were added to 900 μl of a solutioncontaining 84.3 g kg⁻¹ of fatty acid hydroperoxides (including about 75%of the substrate 13-HPOT) previously produced with linseed oilhydrolysate as a source of fatty acids and ground soybeans as the sourceof the 13-lipoxygenase. The reaction was carried out at room temperaturefor 5 minutes. An aliquot of 100 μl of the reaction was diluted in 900μl of water containing 2-3 mg of the reducing agent NaBH₄ and stirredfor 10 minutes. The reduced reaction mixture was extracted with onevolume of ethyl acetate and assayed by gas chromatography for thepresence of 3-(Z)-hexen-1-ol. The consumption of the fatty acidhydroperoxides was followed by HPLC. The molar yield is expressed as theratio between the amount of 3-(Z)-hexen-1-ol obtained during thereaction and the amount of 3-(Z)-hexen-1-ol one could theoreticallyobtain from the amount of 13-HPOT present in the reaction.

The results of this experiment are listed in Table 3, showing theimproved enzymatic activity of the modified lyases compared to therecombinant wild-type enzyme (pWT).

TABLE 3 Activity of modified hydroperoxide lyases NaBH₄ was used as thereducing agent. 3-(Z)-hexen-1-ol Biocatalyst Molar 13HPOT concentrationyield conversion Plasmid (OD₆₀₀) (g l⁻¹) (%) (%) pB7#A 2 7.4 49 59 pAC#52 7.9 53 54 pC2#A 2 9.7 65 55 pE8#B 2 8.8 59 65 pGC#7 2 8.5 57 66 p4E#102 6.4 43 54 p9D#3 2 5.4 36 55 pD10#A 1 0.9 6 N/D* pWT 2 0.8 5 29 *NoData

Experiment 3

E. coli MC1061 cells transformed with the plasmids pB7#A, pAC#5, pC2#A,pE8#B, pGC#7, pD10#A and pWT of Table 1 were cultivated in shake flaskcultures in 100 ml of LB medium with 100 ppm of ampicillin at 180 rpmand at 20° C. for 30 hours without induction. Cultures were centrifuged,the pellet was re-suspended in 100 mM phosphate buffer pH 7.6 and theoptical density was adjusted to OD₆₀₀=10. The catalytic activity wasdetermined as follows: 500 mg of dried baker's yeast were added to 800μl of 100 mM phosphate buffer at pH 7.6 and stirred for 30 seconds. 3.7ml of a solution containing 83 g kg⁻¹ of fatty acid hydroperoxidespreviously produced (including about 75% of the substrate 13-HPOT) withlinseed oil hydrolysate as a source of fatty acids and ground soybeansas the source of the 13-lipoxygenase were added. 500 μL (10% v/v) ofrecombinant cells of E. coli (OD₆₀₀=10) harboring the plasmid ofinterest were then rapidly added under stirring. The reaction was keptat room temperature for 1 hour. An aliquot of 100 μl of the reaction wasdiluted with 900 μl of water, extracted with 1 ml of ethyl acetate andassayed by gas chromatography for the presence of 3-(Z)-hexen-1-ol.

The results of this experiment are listed in Table 4, showing theimproved enzymatic activity of the modified lyases of the presentinvention compared to the recombinant wild-type enzyme (pWT).

TABLE 4 Activity of modified hydroperoxide lyases Baker's yeast servedas the reducing agent. Biocatalyst concentration 3-(Z)-hexen-1-olPlasmid (OD₆₀₀) (g l⁻¹) pB7#A 1 1.2 pAC#5 1 1.2 pC2#A 1 1.5 pE8#B 1 1.1pGC#7 1 2.4 pD10#A 1 0.5 pWT 1 0.2

Experiment 4

E. coli MC1061 cells harboring the plasmids pGC#7, p9D#3, pD10#A and pWTof Table 1 were cultivated in shake flask cultures in 25 ml of LB mediumcontaining 100 ppm of ampicillin at 180 rpm and at 20° C. for 30 hourswithout induction. Cultures were centrifuged and the pellet re-suspendedin 100 mM phosphate buffer pH 7.6. Cell suspensions were prepared havingdifferent optical densities ranging between OD₆₀₀=20 to OD₆₀₀=250. Thecatalytic activity was determined as follows: 100 μl (10% v/v) of cellsuspension (OD₆₀₀=20 to OD₆₀₀=250) were added to 900 μl of a solutioncontaining 84.3 g kg⁻¹ of fatty acid hydroperoxides previously producedwith linseed oil hydrolysate as a fatty acid source and ground soybeansas the source of the 13-lipoxygenase. The reaction was carried out atroom temperature for 5 minutes. An aliquot of 10 μl of the reaction wasdiluted in 990 μl of ethanol and assayed for the consumption of 13HPOTby HPLC analysis. The results of this experiment are shown in FIG. 2.This experiment confirms that the modified 13-hydroperoxide lyases ofthe present invention lead to a 100% conversion of the substrate13-hydroperoxy-octadecatrienoic acid, while the wild-type13-hydroperoxide lyase from guava only converts about 30% of the13-hydroperoxy-octadecatrienoic acid even at an OD₆₀₀ of 25.

Example 4

Performance of the Modified 13-hydroperoxide Lyase GC#7

Experiment 1

Cells of E. coli MC1061:pGC#7 were grown at 20° C. in 2 liter of LBmedium containing 100 ppm of ampicillin in a stirred lab-scale reactorfor 30 h. The culture was aerated with air at 1 vvm. Cells wereharvested by centrifugation and re-suspended in 100 mM phosphate bufferpH 7.6 to yield an optical density of OD₆₀₀=68. Fatty acidhydroperoxides were produced with linseed oil hydrolysate as a source offatty acids and ground soybeans as the source of the 13-lipoxygenase.172 ml of recombinant E. coli cells MC1061:pGC#7 (OD₆₀₀=68) were addedto 1544 g of fatty acid hydroperoxide solution containing 87 g kg⁻¹ ofHPOT/D under stirring at room temperature. The reaction was stoppedafter 5 minutes by adding 50 ml of an aqueous solution containing 36% ofthe reducing agent NaBH4. 3-(Z)-hexen-1-ol was subsequently isolated bydistillation under reduced pressure yielding 8.7 gram of3-(Z)-hexer-1-ol.

Experiment 2

Cells of E. coli MC1061:pGC#7 were grown at 30° C. using a mineral saltmedium containing 100 ppm of ampicillin. The pH was kept at pH 7 and theculture was aerated with air at 1 vvm. The gene expression was inducedby adding 0.5 mM of IPTG at an OD₆₀₀=4. After 24 hours an opticaldensity of OD₆₀₀=9 was reached. Cells were harvested by centrifugationand re-suspended in 100 mM of phosphate buffer of pH 7.6 to give anoptical density of OD₆₀₀=10. 100 μl (10% v/v) of this cell suspensionwere added to 900 μl of a solution containing 83 g kg⁻¹ of fatty acidhydroperoxides previously produced with linseed oil hydrolysate as afatty acid source and ground soybeans as the source of the13-lipoxygenase. The reaction was carried out at room temperature for 5minutes. An aliquot of 100 μl of the reaction mixture was diluted with900 μl of water containing 2-3 mg of the reducing agent NaBH₄. Thereduced reaction mixture was extracted with one volume of ethyl acetateand assayed by gas chromatography for the volatile C₆ alcohols. Aconcentration of 9.5 g l⁻¹ of 3-(Z)-hexen-1-ol was estimated to beproduced by the recombinant MC1061:pGC7 in a reaction as describedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of amino acid sequences of different modified13-hydroperoxide lyases designated as GC7, E8B, B7A, C2A, AC5, D10A,4E10 and 9D3 with the wild-type amino acid sequence according to SEQ IDNo.1. The amino acid insertions and substitutions are shown in boldletters. The first 9 amino acids of variants GC7, E8B, B7A, C2A, AC5,D10A, 4E10 and 9D3 as depicted in SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14 and16 are not shown in the alignment, as they are derived from9-hydroperoxide lyase and not from SEQ ID No. 1.

FIG. 2 shows the consumption of 13-HPOT at different cell densities ofthe E. coli clones expressing the variant or the wild-type13-hydroperoxide lyase, respectively. pGC#7: circles; p9D#3: triangles;pD10#A: squares and pWT: diamonds.

What is claimed is:
 1. A modified 13-hydroperoxide lyase polypeptidecomprising an amino acid sequence which has no more than 40 amino acidalterations as compared to the amino acid sequence of the wild-typeprotein having the amino acid sequence of SEQ ID NO. 1 wherein at leastone amino acid alteration position is selected from the group consistingof positions 3, 4, 5, 19, 208, 340, 342, 352, 354, 358, 359, 360, 371,372, 375, 377, 382, 383, 387, 388, 389, 392, 393, 394, 395, 399 and 457, with said modified 13-hydroperoxide lyase having increased enzymaticactivity as compared to the wild-type protein having the amino acidsequence of SEQ ID NO. 1 and wherein the modified lyase comprises one ormore substitutions selected from the group consisting of V3L, R4K, TSP,519L, N208H, F340L, Y342F, K352R, K352S, H354Y, F358Y, D359E, V360l,K371 P, V372L, T375R, P377S, E382D, P383A, N387K, S388A, D389E, V392M,Q393G, N394E, D399S, D399N and N457K wherein the modified polypeptidehas the amino-terminal amino acid sequence ATPSSSSPE (SEQ ID NO:18). 2.A method for preparing a modified 13-hydroperoxide lyase polypeptidecomprising: culturing one or more transgenic cells transformed with arecombinant nucleic acid molecule comprising a nucleotide sequenceencoding the modified 13-hydroperoxide lyase of claim 1 under conditionswhich permit expression of the polypeptide, and optionally recoveringthe polypeptide.
 3. A method of producing an aldehyde, which comprises:contacting a 13-hydroperoxide of a polyunsaturated fatty acid selectedfrom the group consisting of linoleic acid or alpha-linolenic acid, witha modified 13-hydroperoxide lyase according to claim 1 to produce analdehyde; and recovering the aldehyde.
 4. The method according to claim3, wherein the polyunsaturated fatty acid is linoleic acid oralpha-linolenic acid; and the method further comprises producing the13-hydroperoxide by the activity of a 13-lipoxygenase.
 5. The methodaccording to claim 3, which further comprises reducing the aldehyde tothe corresponding alcohol and optionally recovering the alcohol.